1. Field of the Invention
The present invention relates generally to the protection of plants against phyto-pathogens. In one aspect it provides transgenic plants which have enhanced resistance to phytopathogens. It further provides methods for protecting plants against phytopathogens.
2. Description of the Relevant Art
Living organisms possess several inherent defense mechanisms to fight microbial pathogens. One of the chronologically earliest responses to invasion is a respiratory burst that produces two reactive oxygen species that are microbicidal: superoxide and hydrogen peroxide (Mehdy, M. C. 1994. Plant Physiol. 105: 467-472; Wojtaszek, P. 1997. Biochem. J. 322: 681-692). Hydrogen peroxide and its products from reactions with endogenous metabolites possess relatively moderate antimicrobial activity (Doke et al. 1991. Active Oxygen/Oxygen Stress and Plant Metabolism, E. Pell et al. (Eds.). Am. Soc. Plant Physiol., Rockville Md.). Deadlier antimicrobial compounds such as hypohalites are generated from hydrogen peroxide in some bacterial organisms that contain haloperoxidases, for example, chloroperoxidases. Animals contain myeloperoxidase; and in the presence of myeloperoxidase, animal cells, too, can convert hydrogen peroxide to a much stronger antimicrobial compound, hypochlorous acid (HOCl), i.e.,
H2O2+HClxe2x86x92H2O+HOClxe2x80x83xe2x80x83(1)
Plants routinely become infected by fungi, viruses, and bacteria; however, vascular plants lack haloperoxidases such as myeloperoxidase and consequently lack this potent system of antimicrobial and antifungal protection. Although plants lack this endogenous haloperoxidase-based method of defense, plant fungi are sensitive to the effects of these haloperoxidases in experiments in vitro. Jacks et al. (1991. Biochem. Biophys. Res. Commun. 178: 1202-1204) have shown that the conversion of hydrogen peroxide to hypochlorite, catalyzed by myeloperoxidase, results in as much as a 90-fold increase in lethality against the plant fungus Aspergillus flavus in vitro, suggesting the possibility that plants transformed with a haloperoxidase gene could possess the advantage of an increased disease resistance to phytopathogens. However, for a transformation strategy involving the enzymes, myeloperoxidase or most haloperoxidases, to be successful, the addition of a plethora of genes coding for the multi-component synthesis of each species specific heme-containing prosthetic group is a necessary requirement (Pfeifer et al. 1992. J. Gen. Microbiol. 138: 1123-1131; van Pee, K-H. 1996. Annu. Rev. Microbiol. 50: 375-399) because these heme derivatives are not available in plants.
Accordingly, this invention provides an alternative strategy, specifically, transformation with a gene encoding a functional yet heme-free haloperoxidase. Such a strategy therefore bypasses the need for including nucleic acid sequences which encode additional components related to specific heme groups.
Presently, plant breeding and biocontrol programs, as well as the development of new antimicrobial agents and fungicides, are disease preventive methods that are being successfully practiced to protect crops. However, it is well known that rapid genetic changes take place in pathogenic organisms. Such evolutionary changes result in the resistance genes of the host quickly becoming ineffective. These facts, together with cost, time considerations, and actual field results, necessitate the constant search for novel methods and reagents for protecting agricultural crops.
We have discovered that the expression of a gene encoding a nonheme haloperoxidase in a plant provides protection to the plant against phytopathogens.
In accordance with this discovery, it is an object of the invention to provide a plant transformed with a gene which encodes a nonheme haloperoxidase and which is expressed to confer protection to the plant against phytopathogens. In particular, a plant in which a nonheme chloroperoxidase is expressed is provided.
Another object of the invention is to provide a method of transforming a plant with a gene capable of expressing a nonheme haloperoxidase sufficient to confer protection to the plant against phytopathogens. A method of transforming a plant with a gene encoding a nonheme chloroperoxidase is particularly provided.
Plant transformation vectors comprising the gene which encodes the nonheme haloperoxidase of the invention are also provided, as are plant cells transformed by these vectors, and plants and their progeny containing the genes.
It is an additional object of the invention to provide a host cell containing the DNA of the invention, wherein said host cell is a bacterial cell, in particular, an Agrobacterium tumefaciens cell.
Still further, it is an object of the invention to engineer the DNA molecules of the invention to allow in the plant for constitutive expression, expression in response to an inducer, expression in particular tissues, and expression at particular stages of development.